Metabolic pathways for glucose and fructose: I synthesis and metabolism of fructose by ovine conceptuses.

Fructose, the most abundant hexose sugar in fetal fluids and blood of sheep and other ungulates and cetaceans, is synthesized from glucose via the polyol pathway in trophectoderm and chorion. However, the cell-specific and temporal expression of enzymes for the synthesis and metabolism of fructose in sheep conceptuses (embryo and placental membranes) and placentomes has not been characterized. This study characterized key enzymes involved in fructose synthesis and metabolism by ovine conceptuses throughout pregnancy. Day 17 conceptuses expressed mRNAs for the polyol pathway (SORD and AKR1B1) and glucose and fructose metabolism (HK1, HK2, G6PD, OGT, and FBP), but not those required for gluconeogenesis (G6Pase or PCK). Ovine placentomes also expressed mRNAs for SORD, AKR1B1, HK1, and OGT. Fructose can be metabolized via the ketohexokinase (KHK) pathway and isoforms, KHK-A and KHK-C, were expressed in ovine conceptuses from Day 16 of pregnancy and placentomes during pregnancy in a cell specific manner: KHK-A protein was more abundant in trophectoderm and cotyledons of placentomes, while KHK-C protein was more abundant in endoderm of Day 16 conceptuses and chorionic epithelium in placentomes. Expression of KHK mRNAs in placentomes was greatest at Day 30 of pregnancy (P < 0.05), but not different among days later in gestation. These results provide novel insights into the synthesis and metabolism of fructose via the uninhibited KHK pathway in ovine conceptuses to generate ATP via the TCA cycle, as well as substrates for the pentose cycle, hexosamine biosynthesis pathway and one-carbon metabolism required for conceptus development throughout pregnancy.

Metabolic pathways of glucose and fructose: II spatiotemporal expression of genes involved in synthesis and transport of lactate in ovine conceptuses.

Lactate, an abundant molecule in fetal fluids and blood of mammalian species is often overlooked as a metabolic waste product generated during pregnancy. Most of the glucose and fructose consumed by ovine conceptuses is converted to lactate, but proteins involved in lactate metabolism and transport have not been investigated. This study characterized total lactate produced by ovine conceptuses throughout gestation, as well as expression of mRNAs and proteins involved in lactate metabolism. Lactate increased in abundance in the uterine lumen during the preimplantation period and was more abundant than pyruvate. The abundance of lactate in allantoic and amniotic fluids increased with advancing days of gestation and most abundant on Day 125 of pregnancy (P < 0.05). Lactate dehydrogenase (LDH) subunits A (converts pyruvate to lactate) and B (converts lactate to pyruvate) were expressed by conceptuses throughout gestation. Lactate is transported via monocarboxylic acid transporters SLC16A1 and SLC16A3, both of which were expressed by the conceptus throughout gestation. Additionally, the interplacentomal chorioallantois from Day 126 expressed SLC16A1 and SLC16A3 and transported lactate across the tissue. Hydrocarboxylic acid receptor 1 (HCAR1), a receptor for lactate, was localized to the uterine luminal and superficial glandular epithelia of pregnant ewes throughout gestation, and conceptus trophectoderm during the peri-implantation period of gestation. These results provide novel insights into the spatiotemporal profiles of enzymes, transporters, and receptor for lactate by ovine conceptuses throughout pregnancy.

Conserved and divergent features of trophoblast stem cells.

Trophoblast stem cells (TSCs) are a proliferative multipotent population derived from the trophectoderm of the blastocyst, which will give rise to all the functional cell types of the trophoblast compartment of the placenta. The isolation and culture of TSCs in vitro represent a robust model to study mechanisms of trophoblast differentiation into mature cells both in successful and diseased pregnancy. Despite the highly conserved functions of the placenta, there is extreme variability in placental morphology, fetal-maternal interface, and development among eutherian mammals. This review aims to summarize the establishment and maintenance of TSCs in mammals such as primates, including human, rodents, and nontraditional animal models with a primary emphasis on epigenetic regulation of their origin while defining gaps in the current literature and areas of further development. FGF signaling is critical for mouse TSCs but dispensable for derivation of TSCs in other species. Human, simian, and bovine TSCs have much more complicated requirements of signaling pathways including activation of WNT and inhibition of TGFß cascades. Epigenetic features such as DNA and histone methylation as well as histone acetylation are dynamic during development and are expressed in cell- and gestational age-specific pattern in placental trophoblasts. While TSCs from different species seem to recapitulate some select epigenomic features, there is a limitation in the comprehensive understanding of TSCs and how well TSCs retain placental epigenetic marks. Therefore, future studies should be directed at investigating epigenomic features of global and placental-specific gene expression in primary trophoblasts and TSCs.

Characterization of TNSALP expression, localization, and activity in ovine utero-placental tissues†.

Tissue-nonspecific alkaline phosphatase (TNSALP; encoded by ALPL gene) has a critical role in the regulation of phosphate homeostasis postnatally. However, the utero-placental expression of TNSALP and the role in phosphate transport in pregnancy is poorly understood. Estrous cycles of ewes were synchronized, and ewes were euthanized and hysterectomized on Days 1, 9, or 14 of the estrous cycle or bred to fertile rams and euthanized and hysterectomized on Days 9, 12, 17, 30, 50, 70, 90, 110, or 125 of pregnancy. The expression of ALPL mRNA, immunolocalization of TNSALP protein, and quantification and localization of TNSALP enzymatic activity was performed on ovine endometria and placentomes. Day of the estrous cycle did not alter ALPL mRNA expression or enzymatic activity of TNSALP. TNSALP protein localized to uterine epithelial and stromal cells, blood vessels, myometrium, caruncular, and cotyledonary stroma. TNSALP activity was localized to uterine epithelia, blood vessels, caruncular stroma (from Day 70 of gestation), and the apical surface of chorionic epithelia (from Day 50 of gestation). TNSALP protein and activity localized to the apical surface of uterine epithelia during the estrous cycle and in early pregnancy. Endometrial TNSALP enzymatic activity was downregulated on Days 17 and 30 of gestation (P < 0.05). Expression of ALPL mRNA decreased in late gestation in endometria and placentomes (P < 0.05). TNSALP activity peaked in placentomes on Days 70 and 90 of gestation. Collectively, these results suggest a potential role of TNSALP in the regulation of phosphate transport and homeostasis at the maternal-conceptus interface in ruminants.

Regulation of synthesis of polyamines by progesterone, estradiol, and their receptors in uteri of cyclic ewes†.

Progesterone (P4), estradiol (E2), and expression of their receptors (PGR and ESR1, respectively) by cells of the uterus regulate reproductive performance of mammals through effects on secretion and transport of nutrients into the uterine lumen. This study investigated the effect of changes in P4, E2, PGR, and ESR1 on expression of enzymes for the synthesis and secretion of polyamines. Suffolk ewes (n = 13) were synchronized to estrus (Day 0) and then, on either Day 1 (early metestrus), Day 9 (early diestrus), or Day 14 (late diestrus) of the estrous cycle, maternal blood samples were collected, and ewes were euthanized before obtaining uterine samples and uterine flushings. Endometrial expression of MAT2B and SMS mRNAs increased in late diestrus (P < 0.05). Expression of ODC1 and SMOX mRNAs decreased from early metestrus to early diestrus, and expression of ASL mRNA was lower in late diestrus than in early metestrus (P < 0.05). Immunoreactive PAOX, SAT1, and SMS proteins were localized to uterine luminal, superficial glandular, and glandular epithelia, stromal cells, myometrium, and blood vessels. Concentrations of spermidine and spermine in maternal plasma decreased from early metestrus to early diestrus and decreased further in late diestrus (P < 0.05). The abundances of spermidine and spermine in uterine flushings were less in late diestrus than early metestrus (P < 0.05). These results indicate that synthesis and secretion of polyamines are affected by P4 and E2, as well as the expression of PGR and ESR1 in the endometria of cyclic ewes.

Creatine metabolism at the uterine-placental interface throughout gestation in sheep†.

The placenta requires high levels of adenosine triphosphate to maintain a metabolically active state throughout gestation. The creatine-creatine kinase-phosphocreatine system is known to buffer adenosine triphosphate levels; however, the role(s) creatine-creatine kinase-phosphocreatine system plays in uterine and placental metabolism throughout gestation is poorly understood. In this study, Suffolk ewes were ovariohysterectomized on Days 30, 50, 70, 90, 110 and 125 of gestation (n = 3-5 ewes/per day, except n = 2 on Day 50) and uterine and placental tissues subjected to analyses to measure metabolites, mRNAs, and proteins related to the creatine-creatine kinase-phosphocreatine system. Day of gestation affected concentrations and total amounts of guanidinoacetate and creatine in maternal plasma, amniotic fluid and allantoic fluid (P < 0.05). Expression of mRNAs for arginine:glycine amidinotransferase, guanidinoacetate methyltransferase, creatine kinase B, and solute carrier 16A12 in endometria and for arginine:glycine amidinotransferase and creatine kinase B in placentomes changed significantly across days of gestation (P < 0.05). The arginine:glycine amidinotransferase protein was more abundant in uterine luminal epithelium on Days 90 and 125 compared to Days 30 and 50 (P < 0.01). The chorionic epithelium of placentomes expressed guanidinoacetate methyltransferase and solute carrier 6A13 throughout gestation. Creatine transporter (solute carrier 6A8) was expressed by the uterine luminal epithelium and trophectoderm of placentomes throughout gestation. Creatine kinase (creatine kinase B and CKMT1) proteins were localized primarily to the uterine luminal epithelium and to the placental chorionic epithelium of placentomes throughout gestation. Collectively, these results demonstrate cell-specific and temporal regulation of components of the creatine-creatine kinase-phosphocreatine system that likely influence energy homeostasis for fetal-placental development.

Phosphate, calcium, and vitamin D signaling, transport, and metabolism in the endometria of cyclic ewes.

BACKGROUND: Recent evidence suggests important roles for progesterone (P4) and interferon tau in the regulation of calcium, phosphate, and vitamin D signaling in the uteri of pregnant sheep. However, the effects of P4 and estradiol (E2), with respect to the expression of their receptors PGR and ESR1, respectively, in uterine epithelia on mineral signaling during the estrous cycle has not been investigated. Estrous cycles of mature Suffolk ewes were synchronized, prostaglandin F2α was administered, and ewes were observed for estrus (designated as Day 0) in the presence of vasectomized rams. On Days 1, 9, or 14 of the estrous cycle, hysterectomies were performed. RESULTS: 25-hydroxyvitamin D was more abundant in plasma from ewes on Day 14 than Day 1 (P < 0.05). Expression of fibroblast growth factor receptor 2 (FGFR2), a disintegrin and metalloprotease 17 (ADAM17), and parathyroid hormone-related protein (PTHrP) mRNAs was greater in endometria on Day 9 compared to Days 1 and 14 (P < 0.01). Similarly, expression of transient receptor potential cation channel subfamily V member 6 (TRPV6) mRNA was greater in endometria on Day 9 than Day 1 (P < 0.05). ATPase plasma membrane Ca2+ transporting 4 (ATP2B4) and S100 calcium binding protein G (S100G) mRNA expression was greater in endometria on Day 14 than on Days 1 and 9 (P < 0.01). In contrast, endometrial expression of vitamin D receptor (VDR) mRNA was lower on Days 9 and 14 than Day 1 (P < 0.01). Expression of klotho (KL) (P < 0.05) and cytochrome P450 family 24 subfamily A member 1 (CYP24) (P < 0.01) mRNAs was lower on Day 14 than Days 1 and 9. ADAM17, FGF23, CYP2R1, CYP27B1, KL, and VDR proteins immunolocalized to the uterine myometrium, blood vessels, and uterine luminal (LE), superficial glandular (sGE), and glandular (GE) epithelia. S100A9 protein was weakly expressed in the uterine myometrium, LE, sGE, and GE. Immunoreactivity of CYP2R1 and KL proteins in uterine LE and sGE was less on Day 1 than on Days 9 and 14. In contrast, S100G protein was expressed exclusively by GE, and immunoreactive S100G protein was less on Day 9. S100A12 protein localized to stromal cells of the uterine stratum spongiosum and blood vessels, but not by uterine epithelial cells. CONCLUSION: Collectively, these results implicate E2, P4, and PGR in the regulation of phosphate, calcium, and vitamin D signaling in cyclic ewes.

The ovine conceptus utilizes extracellular serine, glucose, and fructose to generate formate via the one carbon metabolism pathway.

Highly proliferative cells rely on one carbon (1C) metabolism for production of formate required for synthesis of purines and thymidine for nucleic acid synthesis. This study was to determine if extracellular serine and/or glucose and fructose contribute the production of formate in ovine conceptuses. Suffolk ewes (n = 8) were synchronized to estrus, bred to fertile rams, and conceptuses were collected on Day 17 of gestation. Conceptuses were either snap frozen in liquid nitrogen (n = 3) or placed in culture in medium (n = 5) containing either: 1) 4 mM D-glucose + 2 mM [U-13C]serine; 2) 6 mM glycine + 4 mM D-glucose + 2 mM [U-13C]serine; 3) 4 mM D-fructose + 2 mM [U-13C]serine; 4) 6 mM glycine + 4 mM D-fructose + 2 mM [U-13C]serine; 5) 4 mM D-glucose + 4 mM D-fructose + 2 mM [U-13C]serine; or 6) 6 mM glycine + 4 mM D-glucose + 4 mM D-fructose + 2 mM [U-13C]serine. After 2 h incubation, conceptuses in their respective culture medium were homogenized and the supernatant analyzed for 12C- and 13C-formate by gas chromatography and amino acids by high performance liquid chromatography. Ovine conceptuses produced both 13C- and 12C-formate, indicating that the [U-13C]serine, glucose, and fructose were utilized to generate formate, respectively. Greater amounts of 12C-formate than 13C-formate were produced, indicating that the ovine conceptus utilized more glucose and fructose than serine to produce formate. This study is the first to demonstrate that both 1C metabolism and serinogenesis are active metabolic pathways in ovine conceptuses during the peri-implantation period of pregnancy, and that hexose sugars are the preferred substrate for generating formate required for nucleotide synthesis for proliferating trophectoderm cells.

Creatine metabolism at the uterine-conceptus interface during early gestation in sheep†.

Ruminant conceptuses that elongate and attach to the uterine luminal epithelium (LE) to establish pregnancy require a large amount of adenosine triphosphate (ATP). The creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system re-generates ATP in dividing and migrating cells such as the conceptus trophectoderm cells. However, little is known about metabolism of Cr within uterine and conceptus tissues in livestock species during early gestation. In this study, Suffolk ewes were ovariohysterectomized on Days 9, 12, 15, 16, 17, 18, 20, or 21 of pregnancy (n = 2-5 animals/per day) to investigate metabolites, mRNAs, and proteins of the Cr-CK-PCr system at uterine-conceptus interface. Amounts of Cr and guanidinoacetate (GA) in uterine flushings increased between Days 12 and 17 of pregnancy. Endometrial expression of mRNAs for GA formation (AGAT), Cr synthesis (GAMT), and Cr/PCr utilization (CKB) was greater on Days 17 and 21 than on Days 9 and 12 of pregnancy. Immunoreactive AGAT was detected in uteri only on Day 21 but not in uteri or conceptuses at earlier days of pregnancy. GAMT, SLC6A8, and CKs were expressed in uterine luminal and glandular epithelia. Immunoreactive CKs (CKB, CKM, and CKMT1) appeared greater on Day 9 than Day 17 of pregnancy. Immunoreactive GAMT and CKs appeared greater in trophectoderm of conceptuses on Day 20 than on Day 15 of pregnancy, whereas the opposite was observed for that of SLC6A8. This study provides insights into cell-, tissue-, and time-specific metabolism of Cr at the uterine-conceptus interface suggesting a role for the Cr-CK-PCr system in ovine conceptus development and implantation.

Ovine conceptus tissue metabolizes fructose for metabolic support during the peri-implantation period of pregnancy†.

Roles of fructose in elongating ovine conceptuses are poorly understood, despite it being the major hexose sugar in fetal fluids and plasma throughout gestation. Therefore, we determined if elongating ovine conceptuses utilize fructose via metabolic pathways for survival and development. Immunohistochemical analyses revealed that trophectoderm and extra-embryonic endoderm express ketohexokinase and aldolase B during the peri-implantation period of pregnancy for conversion of fructose into fructose-1-phosphate for entry into glycolysis and related metabolic pathways. Conceptus homogenates were cultured with 14C-labeled glucose and/or fructose under oxygenated and hypoxic conditions to assess contributions of glucose and fructose to the pentose cycle (PC), tricarboxylic acid cycle, glycoproteins, and lipid synthesis. Results indicated that both glucose and fructose contributed carbons to each of these pathways, except for lipid synthesis, and metabolized to pyruvate and lactate, with lactate being the primary product of glycolysis under oxygenated and hypoxic conditions. We also found that (1) conceptuses preferentially oxidized glucose over fructose (P < 0.05); (2) incorporation of fructose and glucose at 4 mM each into the PC by Day 16 conceptus homogenates was similar in the presence or absence of glucose, but incorporation of glucose into the PC was enhanced by the presence of fructose (P < 0.05); (3) incorporation of fructose into the PC in the absence of glucose was greater under oxygenated conditions (P < 0.01); and (4) incorporation of glucose into the PC under oxygenated conditions was greater in the presence of fructose (P = 0.05). These results indicate that fructose is an important metabolic substrate for ovine conceptuses.

Inhibition of SHMT2 mRNA translation increases embryonic mortality in sheep†.

The one-carbon metabolism (OCM) pathway provides purines and thymidine for synthesis of nucleic acids required for cell division, and S-adenosyl methionine for polyamine and creatine syntheses and the epigenetic regulation of gene expression. This study aimed to determine if serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in the OCM pathway, is critical for ovine trophectoderm (oTr) cell function and conceptus development by inhibiting translation of SHMT2 mRNA using a morpholino antisense oligonucleotide (MAO). In vitro treatment of oTr cells with MAO-SHMT2 decreased expression of SHMT2 protein, which was accompanied by reduced proliferation (P = 0.053) and migration (P < 0.05) of those cells. Intrauterine injection of MAO-SHMT2 in ewes on Day 11 post-breeding tended to decrease the overall pregnancy rate (on Days 16 and 18) compared with MAO-control (3/10 vs. 7/10, P = 0.07). The three viable conceptuses (n = 2 on Day 16 and n = 1 on Day 18) recovered from MAO-SHMT2 ewes had only partial inhibition of SHMT2 mRNA translation. Conceptuses from the three pregnant MAO-SHMT2 ewes had similar levels of expression of mRNAs and proteins involved in OCM as compared with conceptuses from MAO-control ewes. These results indicate that knockdown of SHMT2 protein reduces proliferation and migration of oTr cells (in vitro) to decrease elongation of blastocysts from spherical to elongated forms. These in vitro effects suggest that increased embryonic deaths in ewes treated with MAO-SHMT2 are the result of decreased SHMT2-mediated trophectoderm cell proliferation and migration supporting a role for the OCM pathway in survival and development of ovine conceptuses.

Regulation of Gene Expression by Amino Acids in Animal Cells.

Amino acids have pleiotropic roles in animal biology including protein and glucose synthesis, cellular metabolism, antioxidant reactions, immune enhancers, and inducers or suppressors of gene expression. Recent studies have revealed important roles of amino acids in the regulation of gene expression in animals. Discoveries of cellular amino acid sensors and their mechanistic pathways have broadened our understanding of how the body responds to the deprivation of nutrients and amino acids in particular. Alterations in concentrations of extracellular amino acids can modulate transcription, translation, posttranscriptional modifications, and epigenetic regulation of genes and proteins. Cells have intracellular amino acid sensors, for example, Sestrin2 for leucine and CASTOR2 for arginine, that respond to sufficiency or deficiency in amino acids, thereby inhibiting or activating downstream signals for gene expression, respectively. The sufficiency of an amino acid in cells ensures its binding to cognate sensors and suppression of inhibitors of MTOR, leading to increased global protein synthesis. On the other hand, deprivation of amino acids activates the amino acid response pathway (GCN2-eIF2a-ATF4), leading to increased selective translation of the activating transcription factor 4 (ATF4). Deficiency of an amino acid itself or via the action of ATF4 suppression of MTORC1 activity limits global protein synthesis. ATF4, in response to low concentrations of cellular amino acids, mediates the transcription of groups of genes such as those for amino acid transport and biosynthesis (ASNS, CAT-1, SNAT2), autophagy (ATG3, ATG10, ATG12), and serine-glycine synthesis (PHGDH, PSAT1, PSPH, MTHFD2). Long-term amino acid starvation has a pronounced effect on cells: suppressed expression and translation of genes required for normal cell growth and metabolism and enhanced expression of genes required for cell adaptation and survival. Levels of amino acids also affect the posttranslational modifications of proteins through mechanisms such as acetylation, ADP-ribosylation, disulfide bond formation, glutamylation, and hydroxylation.

Dietary supplementation of alpha-lipoic acid mitigates the negative effects of heat stress in broilers.

Heat stress accounts for substantial economic loss in the poultry industry by altering the health and performance of chickens. Alpha-lipoic acid (ALA) is a water and fat-soluble antioxidant which is readily absorbed from the intestine resulting in maximum bioavailability. Moreover, ALA acts as a coenzyme in glucose metabolism and helps generate other antioxidants. Considering these benefits, we hypothesized that dietary supplementation of ALA would help mitigate heat stress in poultry. A total of 72 Day-old broiler chicks were randomly assigned into three treatment groups: no heat stress (NHS), heat stress with basal diet (HS), and heat stress with alpha-lipoic acid (HS+ALA); each treatment group had 6 replicate pens with 4 birds in each pen (n = 24/group). The allocated birds were raised under standard husbandry practices for 3 weeks. After 21 d, birds in the HS and HS+ALA groups were exposed to heat stress (33°C for 8 hours during the day) for 3 weeks, while the NHS group was reared under normal conditions (22-24°C). The HS+ALA group received a basal finisher diet fortified with ALA (500 mg/kg) during the treatment period (22 to 42 d), while other birds were provided with the basal finisher diet. Weekly body weight and feed intake were recorded. The cecum digesta for volatile fatty acids (VFAs) analysis and 16S rRNA sequencing for the gut microbiota analysis; and the ileum tissue samples for histological and gene expression analyses were collected on d 42. Exposure to heat stress decreased (P<0.05) average daily gain (ADG) and final body weight (FBW) in the HS group compared to the NHS group, the supplementation of ALA improved (P<0.05) ADG and FBW in heat-stressed birds. Furthermore, birds in the HS+ALA group had increased (P<0.05) expression of HSP90, PRDX1, GPX3, SOD2, OCLN, and MUC2 genes and higher (P<0.05) concentrations of major VFAs (acetate, propionate, and butyrate). The dietary ALA supplementation also improved the villus height and villus height to crypt depth ratio in the HS+ALA group. The microbial diversity analysis revealed significant abundance (P<0.05) of beneficial bacteria Lactobacillus and Peptostreptococcaceae in the cecum of the ALA group. These results indicate that dietary ALA supplementation effectively mitigates the negative effects of heat stress in broilers by improving the expression of heat-shock, tight-junction, antioxidants, and immune-related genes in the intestine, improving villus structures, increasing concentration of major VFAs, and enriching the beneficial microbiota.

RNA sequencing-based analysis of the magnum tissues revealed the novel genes and biological pathways involved in the egg-white formation in the laying hen.

BACKGROUND: The mechanism of egg formation in the oviduct of laying hens is tightly controlled; each segment of the oviduct contributes a unique component of the egg. Several genes/proteins are involved in the synthesis of a completely healthy egg. This implies a time- and tissue-specific expression of genes and proteins in the different oviductal segments. We used hens at different physiological stages and time points to understand the transcriptional regulation of egg-white (albumen) synthesis and secretion onto the eggs in the magnum of laying hens. This study used Next-Generation Sequencing and quantitative real-time PCR (qPCR) to detect the novel genes and the cognate biological pathways that regulate the major events during the albumen formation. RESULTS: Magnum tissues collected from laying (n = 5 each at 3 h post-ovulation, p.o. and 15-20 h p.o.), non-laying (n = 4), and molting (n = 5) hens were used for differential gene expression analyses. A total of 540 genes (152 upregulated and 388 down-regulated) were differentially expressed at 3 h p.o. in the magnum of laying hens. Kyoto Encyclopedia of Genes and Genomes pathways analysis of the 152 upregulated genes revealed that glycine, serine, and threonine metabolism was the most-enriched biological pathway. Furthermore, the top two most enriched keywords for the upregulated genes were amino-acid biosynthesis and proteases. Nine candidate genes associated with albumen formation were validated with qPCR to have differential expression in laying, non-laying, and molting hens. Proteases such as TMPRSS9, CAPN2, MMP1, and MMP9 (protein maturation, ECM degradation, and angiogenesis); enzymes such as PSPH, PHGDH, and PSAT1 (amino-acid biosynthesis); RLN3, ACE, and REN (albumen synthesis, secretion and egg transport); and AVD, AvBD11, and GPX3 (antimicrobial and antioxidants) were recognized as essential molecules linked to albumen deposition in the magnum. CONCLUSIONS: This study revealed some novel genes that participate in the signaling pathways for egg-white synthesis and secretion along with some well-known functional genes. These findings help to understand the mechanisms involved in albumen biosynthesis.

Dietary supplementation of dried plum: a novel strategy to mitigate heat stress in broiler chickens.

BACKGROUND: Heat stress is a significant problem in the poultry industry, causing a severe economic loss due to its detrimental effects on chickens' health and performance. Dried plum (DP) is a good source of minerals, vitamins, antioxidants, and phenolic compounds. Studies have suggested that DP has several health benefits, such as maintaining the body's redox system, immune status, and calcium hemostasis. Based on the health benefits of DP, we hypothesized that the dietary supplementation of DP would alleviate the detrimental effects of heat stress on broiler chickens. RESULTS: To test the hypothesis, day-old broiler chicks (n = 72) were randomly allocated to three treatment groups (n = 24/group): no heat stress (NHS), heat stress (HS), and heat stress with dried plum (HS + DP), and reared under standard conditions. The inclusion of 2.5% DP in the feed of the HS + DP group was made during the treatment period, while birds in other groups were provided with a standard finisher diet. After 21 days, birds in the HS and HS + DP groups were exposed to cyclic heat stress conditions (33 °C for 8 h during daytime) for 3 weeks, while those in the NHS group were reared under normal conditions (22-24 °C). Weekly body weight and feed intake were recorded to calculate the average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). Heat stress significantly decreased the final body weight, ADG, ADFI, and increased FCR compared to the NHS group, whereas dietary supplementation of DP significantly improved these growth performance parameters compared to the HS group. Furthermore, supplementation of DP significantly increased the expression of heat shock protein-related genes (HSF1, HSF3, HSP70, and HSP90), antioxidant-related genes (SOD1, SOD2, GPX1, GPX3, PRDX1, and TXN), tight junction-related genes (CLDN1, and OCLN), and immune-related genes (IL4, MUC2) in the ileum as compared to the HS group. The microbiota analysis showed significant enrichment of Bacillales, Christensenellaceae, Bacillaceae, Peptostreptococcaceae, and Anaerotruncus in heat-stressed birds supplemented with DP as compared to the HS group. Further, DP supplementation also significantly increased the concentration of acetate, propionate, and total VFA in the cecal digesta of the HS + DP group as compared to the HS group. CONCLUSION: These findings suggest that DP supplementation effectively improved the growth performances and gut health parameters in the heat-stressed birds. Thus, dried plum can be a potential feed supplement to mitigate heat stress in broiler chickens.

Impact of Heat Stress on Poultry Health and Performances, and Potential Mitigation Strategies.

Heat stress is one of the major environmental stressors in the poultry industry resulting in substantial economic loss. Heat stress causes several physiological changes, such as oxidative stress, acid-base imbalance, and suppressed immunocompetence, which leads to increased mortality and reduced feed efficiency, body weight, feed intake, and egg production, and also affects meat and egg quality. Several strategies, with a variable degree of effectiveness, have been implemented to attenuate heat stress in poultry. Nutritional strategies, such as restricting the feed, wet or dual feeding, adding fat in diets, supplementing vitamins, minerals, osmolytes, and phytochemicals, have been widely studied and found to reduce the deleterious effects of heat stress. Furthermore, the use of naked neck (Na) and frizzle (F) genes in certain breed lines have also gained massive attention in recent times. However, only a few of these strategies have been widely used in the poultry industry. Therefore, developing heat-tolerant breed lines along with proper management and nutritional approach needs to be considered for solving this problem. Thus, this review highlights the scientific evidence regarding the effects of heat stress on poultry health and performances, and potential mitigation strategies against heat stress in broiler chickens and laying hens.

Expression of follistatin is associated with egg formation in the oviduct of laying hens.

The objective of this study was to examine the expression profiles of follistatin (FST) and its associated molecules (MSTN, INHA, INHBB, INHBA, ACVR2A, and ACVR2B) in the oviduct of laying hens at 3 hr and 20 hr post-ovulation (p.o., n = 5; 35 weeks old), molting (n = 5; 60 weeks old), and non-laying (n = 4; 35-60 weeks old) hens and also to localize the FST by using immunohistochemistry assay. Expression of FST was significantly higher (p < .05), and MSTN was lower in the uterus of laying hens around 15-20 hr p.o. (during eggshell formation), however, their expressions in the magnum remain unchanged across different physiological stages of hens. FST was mainly expressed in the luminal and glandular epithelium of the uterine tissues, and their expression intensity was highest in laying hens during the eggshell mineralization. There was a relatively increased expression of INHA in the magnum of laying hens around 3 hr p.o. as compared to non-laying and molting hens. At the same time (3 hr p.o.), there was a significant (p < .05) decrease in the expression of the INHBB, ACVR2A, and ACV2B. These results indicate that follistatin may regulate the differentiation of uterine luminal and glandular epithelium during eggshell biomineralization.

RNA sequencing-based analysis of the laying hen uterus revealed the novel genes and biological pathways involved in the eggshell biomineralization.

Eggshell is the outermost calcified covering of an egg that protects it from microbial invasion and physical damage, and is critical for egg quality. However, understanding of the genes/proteins and the biological pathways regulating the eggshell formation is still obscure. We hypothesized that the transcriptomic analysis of the chicken uteri using RNA-sequencing may reveal novel genes and biological pathways involved in the eggshell biomineralization. RNA-sequence analysis using uteri of laying hens at 15-20 h post-ovulation (layers, n = 3) and non-laying (non-layers, n = 3) hens was carried out. About 229 differentially expressed genes (DEGs) were up-regulated in the layers compared to the non-layers. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Ingenuity Pathway Analysis (IPA) revealed more than ten novel genes and biological pathways related to calcium transport and mineralization in the uterus. Based on the enriched pathways and molecular function analysis, 12 DEGs related to eggshell mineralization were further analyzed in the uteri of layers (3 h and 15-20 h post-ovulation), non-layers and molters using qPCR. Expressions of OC-116 (regulator of mineralization), OTOP2 (modulator of cellular calcium influx), CALCB (intracellular release of Ca-ions), STC2 (increases alkaline phosphatase activity), and ATP2C2 (cellular import of Ca-ions) were significantly higher in the uteri of laying hen at 15-20 h post-ovulation. This study identified the involvement of novel genes and their proposed biological pathways in the regulation of eggshell formation.